Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc(r)) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc(r) genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc(r) genes was quantified by real-time quantitative PCR. To confirm the Tc(r) gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc(r) genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc(r) genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.     

Detection of a common and persistent tet(L)-carrying plasmid in chicken-waste-impacted farm soil

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tc(r)) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tc(r) soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Identification of a new ribosomal protection type of tetracycline resistance gene,tet(36), from swine manure pits

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.     

Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages

In order to study the prevalence and diversity of tetracycline resistant lactic acid bacteria (Tc(r) LAB) along the process line of two different fermented dry sausage (FDS) types, samples from the raw meat, the meat batter and the fermented end product were analysed quantitatively and qualitatively by using a culture-dependent approach. Both the diversity of the tet genes and their bacterial hosts in the different stages of FDS production were determined. Quantitative analysis showed that all raw meat components of both FDS types (FDS-01 and FDS-08) contained a subpopulation of Tc(r) LAB, and that for FDS-01 no Tc(r) LAB could be recovered from the samples after fermentation. Qualitative analysis of the Tc(r) LAB subpopulation in FDS-08 included identification and typing of Tc(r) LAB isolates by (GTG)5-PCR fingerprinting, plasmid profiling, protein profiling and a characterization of the resistance by PCR detection of tet genes. Two remarks can be made when the results of this analysis for the different samples are compared. (i) The taxonomic diversity of Tc(r) LAB varies along the process line, with a higher diversity in the raw meat (lactococci, lactobacilli, streptococci, and enterococci), and a decrease after fermentation (only lactobacilli). (ii) Also the genetic diversity of the tet genes varies along the process line. Both tet(M) and tet(S) were found in the raw meat, whereas only tet(M) was found after fermentation. A possible relationship was found between the disappearing of species other than lactobacilli and tet(S), because tet(S) was only found in lacotocci, enterococci, and streptococci. These data suggest that fermented dry sausages are among those food products that can serve as vehicles for Tc(r) LAB and that the raw meat already contains a subpopulation of these bacteria. Whether these results reflect the transfer of resistant bacteria or of bacterial resistance genes from animals to man via the food chain is difficult to ascertain and may require a combination of cultivation-dependent and cultivation-independent approaches.     

The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tc(r)) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tc(r) genes. Fecal fosmid libraries were functionally screened for Tc(r), and further PCR-screened for specific Tc(r) genes. Tc(r) fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tc(r), although encoded by different genes and organisms. Tc(r) organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tc(r) gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tc(r) was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tc(r) bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual.     

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment.

A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had "empty" integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTC-resistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

Zoo animals as reservoirs of gram-negative bacteria harboring integrons and antimicrobial resistance genes

A total of 232 isolates of gram-negative bacteria were recovered from mammals, reptiles, and birds housed at Asa Zoological Park, Hiroshima prefecture, Japan. Forty-nine isolates (21.1%) showed multidrug resistance phenotypes and harbored at least one antimicrobial resistance gene. PCR and DNA sequencing identified class 1 and class 2 integrons and many beta-lactamase-encoding genes, in addition to a novel AmpC beta-lactamase gene, bla(CMY-26). Furthermore, the plasmid-mediated quinolone resistance genes qnr and aac(6')-Ib-cr were also identified.     

Homology among tet determinants in conjugative elements of streptococci.

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes.     

Antibiotic resistance genes in multidrug-resistant Enterococcus spp. and Streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation

AIMS: In this study, multidrug-resistant bacteria previously recovered from the indoor air of a large-scale swine-feeding operation were tested for the presence of five macrolide, lincosamide and streptogramin (MLS) resistance genes and five tetracycline (tet) resistance genes. METHODS AND RESULTS: Enterococcus spp. (n = 16) and Streptococcus spp. (n =16) were analysed using DNA-DNA hybridization, polymerase chain reaction (PCR) and oligoprobing of PCR products. All isolates carried multiple MLS resistance genes, while 50% of the Enterococcus spp. and 44% of the Streptococcus spp. also carried multiple tet resistance genes. All Enterococcus spp. carried erm(A) and erm(B), 69% carried erm(F), 44% carried mef(A), 75% carried tet(M), 69% carried tet(L) and 19% carried tet(K). All Streptococcus spp. carried erm(B), 94% carried erm(F), 75% carried erm(A), 38% carried mef(A), 50% carried tet(M), 81% carried tet(L) and 13% carried tet(K). CONCLUSIONS: Multidrug resistance among airborne bacteria recovered from a swine operation is encoded by multiple MLS and tet resistance genes. These are the first data regarding resistance gene carriage among airborne bacteria from swine-feeding operations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of multiple resistance genes reported here suggests that airborne Gram-positive bacteria from swine operations may be important contributors to environmental reservoirs of resistance genes.     

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.     

Genetic determinants of antibacterial resistance among nosocomial Escherichia coli, Klebsiella spp., and Enterobacter spp. isolates collected in Russia within 2003-2007

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.     

Characterization of multidrug-resistant Escherichia coli isolates from animals presenting at a university veterinary hospital

In this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection of Escherichia coli isolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1, dfrA1-aadA1, dfrA17-aadA5, dfrA12-orfF-aadA2, bla(OXA-30)-aadA1, aacC1-orf1-orf2-aadA1, dfr7). Class 2 integrons (13.5%) contained the dfrA1-sat1-aadA1 gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected included bla(TEM), cat, floR, aadB, aphA1, strA-strB, sul2, and tet(B), respectively. The bla(CTX-M-2) gene, encoding an extended-spectrum β-lactamase (ESβL), and bla(CMY-2), encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensal E. coli isolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, the bla(CTX-M-2) gene has not previously been reported in Ireland.     

Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs

The antibiotic resistance phenotype and genotype and the integron type were characterized in 58 Salmonella enterica isolates recovered from Bísaro pigs and wild boars (20 S. Typhimurium, 17 S. Rissen, 14 S. Enteritidis and 7 S. Havana). Most S. Typhimurium isolates (15/20 of Bísaro pigs and wild boars) showed ampicillin, chloramphenicol, streptomycin, tetracycline, sulfonamide, and amoxicillin-clavulanic acid resistances. Of the 17 S. Rissen isolates of both origins, 13 were resistant to ampicillin, tetracycline and trimethoprim-sulfamethoxazole. Among the S. Enteritidis isolates of Bísaro pigs, eight were nalidixic acid-resistant and three were sulfonamide-resistant. The tet(A) or tet(G) genes were detected in most tetracycline-resistant isolates. The intI1 gene was identified in 72.5% of S. enterica isolates in which the conserved region 3' of class 1 integrons (qacEΔ1+sul1) was also amplified, whereas none had the intI2 gene. The dfrA12+orfF+aadA2 gene cassette arrangement was found in the variable region of class 1 integrons in 14 S. Rissen isolates. Fifteen S. Typhimurium isolates had two integrons with variable regions of 1000 and 1200 bp that harbored the aadA2 and blaPSE-1 gene cassettes, respectively. In these isolates the floR and tet(G) genes were also amplified, indicative of the genomic island 1 (SGI1). Salmonella Typhimurium and S. Rissen of animal origin frequently show a multi-antimicrobial resistant phenotype, which may have implications in public health.     

Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection

A macroarray system was developed to screen environmental samples for the presence of specific tetracycline (Tc(R)) and erythromycin (erm(R)) resistance genes. The macroarray was loaded with polymerase chain reaction (PCR) amplicons of 23 Tc(R) genes and 10 erm(R) genes. Total bacterial genomic DNA was extracted from soil and animal faecal samples collected from different European countries. Macroarray hybridization was performed under stringent conditions and the results were analysed by fluorescence scanning. Pig herds in Norway, reared without antibiotic use, had a significantly lower incidence of antibiotic resistant bacteria than those reared in other European countries, and organic herds contained lower numbers of resistant bacteria than intensively farmed animals. The relative proportions of the different genes were constant across the different countries. Ribosome protection type Tc(R) genes were the most common resistance genes in animal faecal samples, with the tet(W) gene the most abundant, followed by tet(O) and tet(Q). Different resistance genes were present in soil samples, where erm(V) and erm(E) were the most prevalent followed by the efflux type Tc(R) genes. The macroarray proved a powerful tool to screen DNA extracted from environmental samples to identify the most abundant Tc(R) and erm(R) genes within those tested, avoiding the need for culturing and biased PCR amplification steps.     

Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.     

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet(B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet(B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.